Effects of ectopic decorin in modulating intracranial glioma progression in vivo, in a rat syngeneic model

Given the failure of conventional treatments for glioblastoma, gene therapy has gained interest considerable in recent years. Gliomas are associated with a state of immunosuppression, which appears to be partially mediated by an increase in secretion of transforming growth factor-beta (TGF-beta) from glioma cells. Decorin, a small proteoglycan which can bind to and inactivate TGF-beta, has been successfully used as an antitumor strategy on stably transfected tumor cells and has been shown to cause growth suppression in neoplastic cells of various histological origins. In this paper, we investigated the use of gene therapy to deliver the decorin transgene in a site-specific manner in an experimental model of intracranial gliomas. Our aim was to inhibit the glioma-associated immunosuppressive state, and prolong the survival of tumor-bearing rats. We studied the effects of decorin gene transfer in the rat CNS-1 glioma model. To assess the effect of ectopic expression of decorin on glioma progression in vivo, stably transfected CNS-1 cells expressing decorin were implanted into the brain parenchyma of syngeneic Lewis rats. The rats implanted with CNS-1 cells expressing decorin survived significantly longer than those in the control groups which received CNS-1 cells that did not express decorin (P < .0001). We then investigated whether the survival observed with decorin expressing cells could be mimicked in vivo, using recombinant adenoviruses (RAds) expressing the decorin gene under the control of two different promoters: the human immediate-early cytomegalovirus (h-IE-CMV) and the glial fibrillary acidic protein (GFAP). In vivo results showed that administration of RAd expressing the human decorin under the control of h-IE-CMV promoter has a small, but significant effect in prolonging the survival of experimental tumor bearing rats (P < .0001). Our data indicate that ectopic decorin expression has the potential to slow glioma progression in vivo. Our results also indicate that expression of decorin has to be present in all cells which constitute the intracranial tumor mass for the inhibition of tumor growth and prolongation of the life expectancy of tumor-bearing rats to be effective.     

Progress and challenges in viral vector-mediated gene transfer to the brain

Gene transfer into the brain allows the manipulation of transgene expression in both time and space. Recently developed gene transfer technologies allow transgenes to be expressed in any anatomically, biochemically or functionally distinct group of brain cells. Gene transfer has been used to alter the expression of neurotransmitter receptors, ion channels, signaling proteins, neuronal growth, differentiation and survival factors, and thus to modify brain anatomy, neuron physiology, behavior and pathology. However, challenges remain in making gene therapy a more widespread tool for the treatment of neurological disease. We have identified the following as areas needing development: access and delivery of viral vectors to the brain; diffusion of viral vectors and transgenes throughout large areas of brain tissue; viral vector side effects and toxicity, inflammatory and immune responses to vectors; long-term stable transgene expression; cell type-specific expression of transgenes; and the ability of the experimenter or physician to switch transgene expression 'on' and 'off' at will. In the last year, neuro-gene therapy has shown that brain defects in experimental disease models can be prevented and corrected, and that viral vectors and encoded transgenes can be made to diffuse over larger brain areas. In addition, the cause of vector-induced inflammation and immune responses have begun to be elucidated, so that rational approaches can be developed to avoid these complications. Further improvements in viral vectors will facilitate clinical trials in the near future.     

Subcellular post-transcriptional targeting: delivery of an intracellular protein to the extracellular leaflet of the plasma membrane using a glycosyl-phosphatidylinositol (GPI) membrane anchor in neurons and polarised epithelial cells

The effectiveness of viral vector-mediated gene transfer depends on the expression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribution of expressed transgenes. Targeting individual transgenes to particular subcellular compartments will provide various advantages in increasing the safety, efficacy, and specificity of viral vector-mediated gene delivery. Viruses normally hijack the cellular protein synthesis machinery for their own advantages. It is thus unknown whether cells infected with viral vectors will be able to target proteins to the correct subcellular organelles, or whether the subcellular targeting machinery would be selectively disrupted by viral infection. In this article we explored whether a herpes simplex virus type 1-derived vector could be used to deliver a transgene engineered to be targeted to the extracellular membrane of target cells. To do so we constructed a temperature-sensitive mutant HSV-1 vector, tsK-TT21 expressing a recombinant marker protein, tissue inhibitor of metalloproteinases (TIMP), linked to sequence encoding a signal for the addition of a glycosyl-phosphatidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results demonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, without disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell membrane to modify cell-cell interactions, the function of specific plasma membrane proteins, or their interactions with other membrane proteins, and also to target a prodrug converting enzyme to the plasma membrane of target cells, therefore enhancing its cell killing effects.     

Evaluation of the Sheffield system for identifying children at risk from unexpected death in infancy. Results from Birmingham and Newcastle upon Tyne

The "at birth" system which is used in Sheffield to identify children likely to die unexpectedly in infancy, was tested retrospectively in Birmingham (83 cases) and in Newcastle upon Tyne (56 cases). The discrimination between cases and age-matched controls was poor in both cities. Analysis of the 8 factors used in the system showed that only 2 maintained significant case/control differences in Birmingham and Newcastle. Further investigation showed that other factors from maternity records showed significant case/control differences in these cities. Although the system used in Sheffield would not be of use in a prospective prevention programme in either Newcastle or Birmingham, the possibility of evolving an "at risk" system which might apply more widely is discussed.     

高三尖杉酯碱酰胺的结构测定

三尖杉树皮粗提取物中的一个新生物碱—高三尖杉酯碱酰胺(homoharringtonamide)的结构,经质谱—质谱分析,初步建议为16。类似的酰胺类生物碱,例如三尖杉碱酰胺(cephalotaxamide,6)、11-羟基三尖杉碱酰胺(11-hydroxycephalotaxamide,9)、三尖杉酯碱酰胺(harfingtonamide,14)或异三尖杉酯碱酰胺(isoharringtonamide,15)也可能存在,后三者(9,14,15)尚未见报道。     

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection

Aim: To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS). Methods: Ninety‐nine stools from 38 infants of median 27‐week gestation were cultured; 44 stools from 27 infants had their microbial profiles determined by 16S. Ordination analyses explored effects of patient variables on gut communities. Results: Standard microbiological culture identified a mean of two organisms (range 0–7), DGGE 12 (range 3–18) per patient. Enterococcus faecalis and coagulase negative staphylococci (CONS) were most common by culture (40% and 39% of specimens). Meconium was not sterile. No fungi were cultured. Bacterial community structures in infants with NEC and LOS differed from healthy infants. Infants who developed NEC carried more CONS (45% vs 30%) and less Enterococcus faecalis (31% vs 57%). 16S identified Enterobacter and Staphylococcus presence associated with NEC/LOS, respectively. Conclusions: Important differences were found in the gut microbiota of preterm infants who develop NEC/LOS. The relationship of these changes to current practices in neonatal intensive care requires further exploration.     

One-year expression from high-capacity adenoviral vectors in the brains of animals with pre-existing anti-adenoviral immunity: clinical implications.

The main challenge of gene therapy is to provide long-term, efficient transgene expression. Long-term transgene expression from first generation adenoviral vectors (Advs) delivered to the central nervous system (CNS) is elicited in animals not previously exposed to adenovirus (Ad). However, upon systemic immunization against Ad, transgene expression from a first generation Adv is abolished. High-capacity Advs (HC-Advs) provide sustained very long-term transgene expression in the brain, even in animals pre-immunized against Ad. In this study, we tested the hypothesis that a HC-Adv in the brain would allow for long-term transgene expression, for up to 1 year, in the brain of mice immunized against Ad prior to delivery of the vector to the striatum. In na?ve animals, the expression of beta-galactosidase from Adv or HC-Adv was sustained for 1 year. In animals immunized prior to vector delivery, expression from a first generation Adv was abolished. These results point to a very long-term HC-Adv-mediated transgene expression in the brain, even in animals that had been immunized systemically against Ad before the delivery of HC-Adv into the brain. This study therefore indicates the utility of HC-Adv as a powerful gene therapy vector for chronic neurological disorders, even in patients who had been pre-exposed to Ad prior to gene therapy.